Storing data encoded DNA in living organisms

ABSTRACT

Current technologies allow the generation of artificial DNA molecules and/or the ability to alter the DNA sequences of existing DNA molecules. With a careful coding scheme and arrangement, it is possible to encode important information as an artificial DNA strand and store it in a living host safely and permanently. This inventive technology can be used to identify origins and protect R&amp;D investments. It can also be used in environmental research to track generations of organisms and observe the ecological impact of pollutants. Today, there are microorganisms that can survive under extreme conditions. As well, it is advantageous to consider multicellular organisms as hosts for stored information. These living organisms can provide as memory housing and protection for stored data or information. The present invention provides well for data storage in a living organism wherein at least one DNA sequence is encoded to represent data and incorporated into a living organism.

This invention was made with Government support under Contract DE-AC0676RL01830 awarded by the U.S. Department of Energy. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to a method of storing data. In particular, but not exclusively, the present invention relates to storage of data as encoded DNA in living organisms.

BACKGROUND OF THE INVENTION

A data preservation problem looms large behind today's information superhighway. All current storage (e.g. paper, magnetic media, silicon chips) media require constant attention to maintain their information content. People or natural disasters can easily destroy all of them intentionally or accidentally. With the large amount of information generated by our society every day, it is time to think of a new generation of data memory.

The use of deoxyribonucleic acid (DNA) as a component of memory storage has been proposed for a number of reasons. For example, DNA as a memory medium is compact. One cubic centimeter of DNA in solution could store 10ˆ21 bits of information, whereas, a current conventional computer has a memory of at most 10ˆ14 bits. Also, most computers operate linearly, one block of data after another. Biochemical reactions are highly parallel in operation. That is a single biochemical operation can affect trillions of DNA strands in a test tube.

Heller et. al. (U.S. Pat. No. 5,787,032) describe the use of synthetic DNA polymers as an optical storage media for memory. Clelland et. al. reported in Nature (Vol. 399, 10 Jun. 1999, pp. 533-34 or www.nature.com) that encoding meaningful information as DNA sequences is possible. The authors conducted an experiment wherein an encoded DNA strand was hid behind a period (i.e., a dot) of a printed document. The document was then sealed and mailed to its owners using regular US Postal Service. The embedded message was successfully recovered in a lab environment. This work proved that a DNA strand can be a substitute for a piece of paper in terms of information storage. However, a naked DNA molecule can easily be destroyed when exposed to unfavorable environmental conditions such as excessive temperature or dessication/rehydration. Even nucleases in the environment may degrade the DNA molecules over time. Therefore, exploiting DNA as a memory medium would require an effective protective storage medium.

Establishing memory of stored information in a living organism can provide adequate protection for the encoded DNA strands. By providing a living host for the DNA—one that can tolerate the addition of “artificial” gene sequences and survive extreme environmental conditions. Perhaps more importantly, the host needs to be able to grow and multiply with the embedded information. Propagation of a host for memory embodied in DNA can allow for preservation and continuation of the stored memory, as well as protecting the integrity of the information contained in the memory. As well there is opportunity to utilize this capability to store purposeful information.

SUMMARY OF THE INVENTION

With a careful coding scheme and arrangement, applicants have invented a process to encode data or information as an artificial DNA strand and store it in a living host safely and permanently. The instant invention can be used to identify origins and protect R&D investments (i.e., DNA watermarking) such as agricultural products and rare animals. For example, the present invention allows for storage of data that comprises specific information about the host organism. The agricultural industry can use this invention to “label” crops. By storing various data regarding the particular plant, including origin, type, generation, etc., the agricultural industry can then rely on this information at a later date (e.g., when produce hits the market). It can also be used in environmental research to track generations of organisms and observe the ecological impact of pollutants. Today, there are microorganisms that can survive heavy radiation exposure, high temperatures, and many other extreme conditions. These hardy microorganisms can serve as memory hosts and protect the stored data or information. There are living organisms such as weeds and cockroaches that have existed on earth for hundreds of millions of years. These organisms are excellent candidates as well for preserving critical information for a future civilization.

Therefore, one embodiment of the present invention is a method of storing data in a living organism wherein at least one DNA sequence is encoded to represent data and incorporated into a living organism.

Another embodiment of the present invention is to provide sequences encoded to represent data with other sequences not specifically coded and incorporating them into a living organism for the purpose of memory storage.

Yet another embodiment of the present invention is to provide a method of storing programmed data into a living organism.

Still another embodiment of the present invention is to provide a memory storage system wherein DNA, encoded to represent data, is stored in a living organism.

Yet another embodiment of the present invention is to provide a method of storing editable data in a living organism.

Still another embodiment of the present invention is to provide a method of storing programmed data that responds to a stimulus into a living organism.

Yet another embodiment of the present invention is to provide a method of storing information that responds to a stimulus and reacts to specific encoded programming into a living organism.

Still another embodiment of the present invention is to provide a memory storage system wherein a living organism comprises at least one DNA sequence encoded to represent data, which is incorporated into the native DNA of a living organism.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be readily obtained by reference to the following Description and the accompanying drawings in which like numerals in different figures represent the same structures or elements, wherein:

FIG. 1 is a simplified schematic diagram of the process of present invention.

FIG. 2 is an illustration of a plasmid vector with encoded DNA inserted.

FIG. 3 is a graphical illustration of the encoded DNA sequence and the decoded message.

FIG. 4 shows an example of a DNA sequence of a song phrase.

DETAILED DESCRIPTION

The present invention comprises a method of storing data in a living organism, wherein at least one DNA sequence is encoded to represent data and is thereafter incorporated into the living organism. The method of the present invention comprises encoding DNA to represent specific data by selecting at least 2 of the four DNA nucleotide bases to represent specific text and arranging the nucleotide bases in a manner to represent the data. Encoding the DNA bases to represent specific data can be achieved in numerous and varied ways and the embodiments set forth herein are not meant to be exclusive, but rather exemplify the broader aspects inherent to the present invention.

The present invention comprises a method of storing data in a living organism by incorporating encoded DNA into a viable cell of said living organism. FIG. 1 shows a simplified schematic of one aspect of the present invention. Firstly, the data to be stored is encoded into a DNA sequence. The four-nucleotide bases associated with a DNA sequence are assembled to represent the specific data by correlation with a specific code. For example, three of the four basic nucleotide bases (Cytosine or C, Guanine or G, Thymine or T and Adenine or A) can be assigned to represent a text character. A string of DNA nucleotide bases can then be assembled to represent text information or data. Once the specific data has been encoded into a DNA sequence, it is then inserting into a vector that will provide as a “vehicle” for transport into a living organism. A vector is a DNA molecule originating from a virus, a plasmid, the cell of a higher organism or synthetically assembled, into which another DNA fragment of appropriate size can be integrated without loss of the vectors capacity for self-replication; vectors introduce foreign DNA into host cells, where it can be reproduced in large quantities. Vectors can be selected from the group consisting of plasmids, cosmids, and yeast artificial chromosomes, and recombinant molecules containing DNA sequences. The vector comprising the encoded DNA is then introduced into a viable cell of a living organism. It is understood by those skilled in the art that DNA bases can be incorporated into a living cell in different ways and the particular vectors used and specific methodology is dependant upon the type of host cell. Once the vector is inside the cell of the host living organism, it can reside and be stored indefinitely. The vector DNA, along with the encoded DNA will be regenerated and coexist with the hosts genomic DNA.

The stored data can than be retrieved by means currently know by those skilled in the art. Stored data can be retrieved by standard PCR amplification method as PCR product (DNA fragment). Standard DNA sequencing method such as the dideoxy termination method can then identify the stored information within the amplified PCR product. Alternatively, stored information within the PCR product can be determined by hybridization with a panel of known oligonucleotides. Once the data is retrieved, it is then encoded and the original message is obtained.

Another aspect of the invention is to encode the DNA to represent data that is programmed data. The programmed data can then be programmed to accomplish an activity, continue a communication process, and even respond to a stimulus that will then result in an action. For example, we can construct a gene fusion between a hydrogen-peroxide-inducible promoter with a lysozyme gene, which will kill the bacteria if we add hydrogen peroxide to the engineered bacteria. We can also construct gene fusion with a regulatory gene, which will trigger a cascade of genetic responses (in our case is information). Gene fusions technique is a very common technique that has been used in studying bacterial gene regulation such as the use of green fluorescent protein.

The living organism utilized in the present invention can be single-celled or multi-cellular, prokaryotic or eukaryotic. Although bacterial cells serve well as host organisms to demonstrate the present invention, it is understood that other living cells can be utilized as well.

Another aspect of the present invention is the storage of data in multicellular living organisms. This embodiment of the present invention can be achieved by incorporating at least one DNA sequence encoded to represent data into a germ cell; a precursor cell that gives rise to gametes that will then serve as specialized haploid cells (sperm or egg) in sexual reproduction, or stem cell; a relatively undifferentiated cell that will continue dividing indefinitely, throwing off (producing) daughter cells that will undergo terminal differentiation into particular cell types. The encoded DNA sequence will then propagate into a multicellular living organism. This embodiment of the invention is a memory storage system that takes advantage of multicellular organisms (e.g., insect, rodent) and serves to propagate the encoded DNA sequence in all daughter cells stemming from the original host stem cell.

The present invention comprises a memory storage system wherein a living organism comprises therein at least one DNA sequence encoded to represent data. The stored data resides in a living organism and remains there until recovery is desired. The data is then retrieved and decoded so as to enable communication. Like a computer memory device that can store data and programs, the present invention comprises the same or similar items in a DNA memory system. Unlike a computer compiled software program, a program in a DNA memory system can comprise a set of rules, options, or instructions that respond to specific circumstantial or environmental conditions. In other words, the living organism will detect stimuli condition as well as react according to the information or instructions encoded in the DNA sequence. The host cell of the living organism should not express the non-native encoded DNA (artificial to the genomic DNA of the organism) and cause destructive consequences such as toxic effects. It is desired to custom-design an encoded DNA sequence that will respond to specific events and cause the host cell of the living organism to react or change. Therefore, the present invention provides a unique nano-scaled event detection tool that will detect and respond to a plurality of stimuli based on the programming encoded into the DNA that is incorporated into a host cell of a living organism.

For a clear and concise understanding of the specification and claims, including the scope given to such terms, the following definitions are provided:

As used herein, the word ENCODE means to express given data or information by means of a code.

As used herein, the word DATA means Information of any form that is used for communication, analysis, and or reasoning in making decisions.

Cells to be used as a carrier of the encoded DNA needs to be made competent using standard methods and will uptake the encoded DNA molecules. This can be achieved by either chemical transformation or electroporation methods.

EXAMPLE 1

DNA Host Identification—Two well-understood bacteria, Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans), were utilized for our experiment. We selected E. coli and D. radiodurans because microorganisms, in general, grow very rapidly and the embedded information can be inherited rapidly and continuously. Deinococcus, survive extreme conditions such as ultraviolet, desiccation, partially vacuum environments, and ionizing radiation up to 1.6 million Rad (about 0.1% of the same radiation dose would be fatal to human beings). Some strains of Deinococcus can also tolerate high temperature. Although bacteria were chosen as preferred embodiments, it is understood that any living cell, whether single-celled or multicellular organism, can be used in the use of this invention.

Information Encoding—A (Adenine), C (Cytosine), G (Guanine), and T (Thymine) were used to assemble a DNA sequence information stream to represent data. Table 1 depicts the encoding key for a set of triplets—a DNA sequence with any 3 of the 4 basic units. It is recognized that other types and methods of coding information can be utilized and this example is not meant to be exclusive to this invention. TABLE 1 DNA encoding table AAA - 0 AAC - 1 AAG - 2 AAT - 3 ACA - 4 ACC - 5 ACG - 6 ACT - 7 AGA - 8 AGC - 9 AGG - A AGT - B ATA - C ATC - D ATG - E ATT - F CAA - G CAC - H CAG - I CAT - J CCA - K CCC - L CCG - M CCT - N CGA - O CGC - P CGG - Q CGT - R CTA - S CTC - T CTG - U CTT - V GAA - W GAC - X GAG - Y GAT - Z GCA - SP GCC - : GCG - , GCT - - GGA - . GGC - ! GGG - ( GGT - ) GTA - {grave over ( )} GTC - ‘ GTG - “ GTT - ” TAA - ? TAC - ; TAG - / TAT - [ TCA - ] TCC - TCG - TCT - TGA - TGC - TGG - TGT - TTA - TTC - TTG - TTT -

Unique DNA Searching—The entire genomic sequence of E. coli and D. radiodurans are known. A number of fixed-size sequences (20-base pairs) were identified. Several criteria were used to identify these set of 20-mers−1. these sequences do not exist in either Deinococcus radioduran or Escherchia coli genome; 2. the 20-mer will not have complimentary sequence with more than four bases at the 3′ end, e.g.-AATT or -CCGG at 3′ end; 3. the GC content of the 20-mer will be in the range of 40 to 60%; 4. the 20-mer will have at least any two of TAG, TAA or TGA stop codons. Criteria 1 to 3 will provide unique tags for subsequent PCR retrieval of encoded DNA, while criterion 4 will prevent the formation of fusion proteins that may be detrimental to the host bacterium. These sequences ensure that no unnecessary mutations or damage to the bacteria result. The sequences will serve as sentinels to tag the beginning and end of the embedded messages—similar to the file header and footer in a magnetic tape—for later identification and retrieval. Of the 10 billion potential candidates in the bacterium Deinococcus, we found only 25 qualified sequences that are acceptable for our experiments. A brutal force computational analysis is used to compute this set of 20-mers. There is 420 combinations of 20-mers. All the 20-mers do not have GC content (% of G or C within the 20-mer) between 40 to 60% were eliminated, and then the 4.1 million 20-mer exist in Deinococcus radiodurans were eliminated. Finally, sequences with complementary 3′ end (-AATT,-TTAA,-GGCC,-CCGG,-ATAT,-TATA,-GCGC,-CGCG), SEQ ID.: 1 were eliminated. The remaining 20-mer was searched for the presence of stop codons. The sequences shown in Table 2 are the identified DNA sequences used to design oligonucleotides (oligos) used herein. Multiple stop codons (i.e., triplets such as TAA, TGA, and TAG) are present in many of the sequences. These codons discourage the host from “reading” the non-native DNA that has been encoded to represent data, and subsequently producing chimeric proteins that may be harmful to the bacteria. TABLE 2 25 20-base Pair Sequences Utilzed Herein. SEQ ID NO.: 2 AAGGTAGGTAGGTTAGTTAG SEQ ID NO.: 3 AGGTTTGGTGGTATAGTTAG SEQ ID NO.: 4 ATAGGAGTGTGTGTAGTTAG SEQ ID NO.: 5 ATATTAGAGGGGGTAGTTAG SEQ ID NO.: 6 GGAGTAGTGTGTATAGTTAG SEQ ID NO.: 7 GGGAGTATGTAGTTAGTTAG SEQ ID NO.: 8 GGTTAGATGAGTGTAGTTAG SEQ ID NO.: 9 TAAGGGATGTGTGTAGTTAG SEQ ID NO.: 10 TAGAGGAGGGATATAGTTAG SEQ ID NO.: 11 TAGATGGGAGGTATAGTTAG SEQ ID NO.: 12 TAGGAGAGATGTGTAGTTAG SEQ ID NO.: 13 TATAGGGAGGGTATAGTTAG SEQ ID NO.: 14 TGTGGGATAGTGATAGTTAG SEQ ID NO.: 15 AGAGTAGTGAGGATAGTTAG SEQ ID NO.: 16 ATAAGTAGTGGGGTAGTTAG SEQ ID NO.: 17 ATAGGGGTATGGATAGTTAG SEQ ID NO.: 18 ATGGGTGGATTGATAGTTAG SEQ ID NO.: 19 GGGAATAGAGTGTTAGTTAG SEQ ID NO.: 20 GGGATGATTGGTTTAGTTAG SEQ ID NO.: 21 GTATGGGAATGGTTAGTTAG SEQ ID NO.: 22 TAGAGAGAGTGTGTAGTTAG SEQ ID NO.: 23 TAGAGTGGTGTGTTAGTTAG SEQ ID NO.: 24 TAGATTGGATGGGTAGTTAG SEQ ID NO.: 25 TAGGGTTGGTAGTTAGTTAG SEQ ID NO.: 26 TATAGGGTAGGGTTAGTTAG

Laboratory Procedures and Results

Two 46-mer complementary oligos were created, each comprising two different 20-mer oligos connected by a 6-base pair long restriction enzyme site. The two 20-mer oligos were created from two different sequences listed in Table 2. The restriction enzyme site was to prepare for later encoded DNA fragment insertion. These two 46-mer long complementary oligos form a double stranded 46-base pair DNA fragment. The DNA fragment was then cloned into a recombinant plasmid as illustrated in FIG. 3 where two 20-mer long oligos 104 serve as “sentinels placed at the beginning and end of the inserted encoded DNA 102, which was then incorporated into plasmid vector 100. Because the two 20-mer oligos do not exist in the genome of the host, they served as identification markers for later message retrieval. The stop codons in these two oligos also help protect the message as well as the host from any potential damage. Table 3 shows the phrases considered for insertion, along with their respectively coded sequences. For this experiment, each phrase used (2, 3, 4, 5, 8, 9, 11) was inserted into a different single cell of D. radians. The present invention can be practiced such that all of the desired phrases are inserted into the same single cell or individual phase can be inserted into different cell. Two complimentary oligos

(5′AGAGTAGTGAGGATAGTTAGAGATCTCTCTAATCACACACATCTCA3′, SEQ ID NO.: 27 and 5′TGAGATGTGTGTGATTAGAGAGATCTCTAACTATCCTCACTACTCT3′, SEQ ID NO.: 28 containing two arbitrarily chosen 20-mer tags (5′AGAGTAGTGAGGATAGTTAG3′, SEQ ID NO.: 29 5′TGAGATGTGTGTGATTAGAG31), SEQ ID NO.: 30 arbitrarily selected from Table 2, were chemically synthesized. These two chemically synthesized oligos (46-mer) were allowed to anneal to each other to form a 46 bp DNA fragment, which was cloned into a cloning vector, pCR-blunt (InVitrogen Inc.). A BglII restriction enzyme site, AGATCT, was built in within 46 bp DNA fragment. As a result, encoded DNA message can be cloned into the BlgII site by standard cloning procedure, and the message can be retrieved with that pairs of tags or primer pairs present within the plasmid vector. (See FIG. 2) TABLE 3 Stored Data Utilized Herein 1 A WORLD OF TEARS, 2 AND A GOLDEN SUN, 3 AND A SMILE MEANS 4 AND A WORLD OF FEARS, 5 AND THE OCEANS ARE WIDE, 6 FRIENDSHIP TO EVERYONE, 7 IT IS TIME WE'RE AWARE. 8 ITS A SMALL SMALL WORLD. 9 IT'S A SMALL WORLD AFTER ALL, 10 IT'S A WORLD OF HOPES 11 IT'S A WORLD OF LAUGHTER, 12 IT'S SMALL SMALL WORLD. 13 THERE IS JUST ONE MOON 14 THERE'S SO MUCH THAT WE SHARE, 15 THOUGH THE MOUNTAINS ARE HIGH,

1 AACGCAAGGGCAGAACGACGTCCCATCGCACGAATTGCACTCATGAGGCGTCTAGCG SEQ ID NO.: 31 2 AAGGCAAGGCCTATCGCAAGGGCACAACGACCCATCATGCCTGCACTACTGCCTGCG SEQ ID NO.: 32 3 AATGCAAGGCCTATCGCAAGGGCACTACCGCAGCCCATGGCACCGATGAGGCCTCTA SEQ ID NO.: 33 4 ACAGCAAGGCCTATCGCAAGGGCAGAACGACGTCCCATCGCACGAATTGCAATTATG SEQ ID NO.: 34 AGGCGTCTAGCG 5 ACCGCAAGGCCTATCGCACTCCACATGGCACGAATAATGAGGCCTCTAGCAAGGCGT SEQ ID NO.: 35 ATGGCAGAACAGATCATGGCG 6 ACGGCAATTCGTCAGATGCCTATCCTACACCAGCGCGCACTCCGAGCAATGCTTATG SEQ ID NO.: 36 CGTGAGCGACCTATGGCG 7 ACTGCACAGCTCGCACAGCTAGCACTCCAGCCGATGGCAGAAATGGTCCGTATGGCA SEQ ID NO.: 37 AGGGAAAGGCGTATGGGA 8 AGAGCACAGCTCGTCCTAGCAAGGGCACTACCGAGGCCCCCCGCACTACCGAGGCC SEQ ID NO.: 38 CCCCGCAGAACGACGTCCCATCGGA 9 AGCGCACAGCTCGTCCTAGCAAGGGCACTACCGAGGCCCCCCGCAGAACGACGTCC SEQ ID NO.: 39 CATCGCAAGGATTCTCATGCGTGCAAGGCCCCCCGCG 10 AACAAAGCACAGCTCGTCCTAGCAAGGGCAGAACGACGTCCCATCGCACGAATTGCAC SEQ ID NO.: 40 ACCGACGCATGCTA 11 AACAACGCACAGCTCGTCCTAGCAAGGGCAGAACGACGTCCCATCGCACGAATTGCAC SEQ ID NO.: 41 CCAGGCTGCAACACCTCATGCGTGCG 12 AACAAGGCACAGCTCGTCCTAGCACTACCGAGGCCCCCCGCACTACCGAGGCCCCCCG SEQ ID NO.: 42 CAGAACGACGTCCCATCGGA 13 AACAATGCACTCCACATGCGTATGGCACAGCTAGCACATCTGCTACTCGCACGACCTATG SEQ ID NO.: 44 GCACCGCGACGACCT 14 AACACAGCACTCCACATGCGTATGGTCCTAGCACTACGAGCACCGCTGATACACGCACTC SEQ ID NO.: 45 CACAGGCTCGCAGAAATGGCACTACACAGGCGTATGGCG 15 AACACCGCACTCCACCGACTGCAACACGCACTCCACATGGCACCGCGACTGCCTCTCAG SEQ ID NO.: 46 GCAGCCTCTAGCAAGGCGTATGGCACACCAGCAACACGCG

The embedded DNA (Table 3) was then inserted into a plasmid vector 100, shown in FIG. 3. The resultant vectors are then transferred into 10 E. coli by electroporation (high-voltage shocks). It is recognized by one of ordinary skill in the art to transfer vectors by other means that may be more particularly suited for the specific host cell. For example, we have used pCRblunt for cloning most of the specifically designed oligos. As bacteria grow and divide, the recombinant plasmid vectors also replicate to produce an enormous number of copies of DNA plasmid vectors containing the encoded DNA. This produces multiple copies of the encoded DNA fragment, allowing storage and continuation of the stored data.

The stored data was then recovered by searching for the two 20-mer oligos (data markers) 104 (FIG. 3). The cells were harvested then lysed to obtain crude genomic DNA comprising the incorporated encoded DNA. With standard procedures, the encoded DNA was located and amplified with polymerase chain reaction (PCR) techniques. Specific primers (M13 reverse, TGAGCGGATAACAATTTCACACAG, SEQ ID NO.: 48 or M13 sequencing primer, GTTTTCCCCAGTCACGACGTTG), SEQ ID NO.: 49 or a pair of tag primers (FIG. 2) can be used to amplify the encoded DNA as PCR DNA fragment. (You might want to add some detail and technically bolster this KK) Once the encoded information was obtained, it was then decoded to reveal the original data (song phrases). FIG. 4 shows an example of a DNA sequence of a song phrase recovered and the decoded message revealed. We use a simple script to convert DNA sequence into words based on our assignment of each of the triplets. We have data from E. coli only although we have tried once with D. radiodurans but not successful yet.

Although the invention has been described with respect to specific preferred embodiments, many variations and modifications may become apparent to those skilled in the art. It is therefore the intention that the appended claims be interpreted as broadly as possible in view of the prior art to include all such variations and modifications. 

1-34. (canceled)
 35. A living organism, comprising: a. DNA encoded to represent data to be decoded thereafter.
 36. A living organism as in claim 35, wherein said organism is a single-celled organism.
 37. A living organism as in claim 36, wherein said single-celled organism is a bacterial cell.
 38. A living organism as in claim 37, wherein said bacterial cell is Deinococcus radians.
 39. A living organism as in claim 37, wherein said bacterial cell is a Escherichia coli. 